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Oral vaccination of calves with an aromatic-dependent Salmonella dublin (O9,12) hybrid expressing O4,12 protects against S. dublin (O9,12) but not against Salmonella typhimurium (O4,5,12).

Three groups of six calves each, 5 to 7 weeks old, were orally vaccinated with the live aromatic-dependent delta aroA Salmonella dublin (O9,12) hybrid strain SL7103 with the O4,12-specifying rfb gene cluster from Salmonella typhimurium. SL7103 was given in three weekly doses, increasing from 2 x 10(9) to 1 x 10(11) bacteria per ml, was well tolerated, and caused mild, short-term temperature increases which diminished with each immunization. The strain was shed for up to 1 week. Strain SL7103 elicited significant (P < 0.001) and equal anti-S. dublin and -S. typhimurium lipopolysaccharide serum antibody responses and skin delayed-type hypersensitivity immune responses. Six vaccinated calves orally challenged with 10(10) CFU (equivalent to 1,000 50% lethal doses) of the virulent parent strain S. dublin SVA47 were protected and experienced only transient fever and mild mucoid diarrhea. However, six vaccinated calves orally challenged with 3 x 10(9) CFU and another six challenged with 3 x 10(8) CFU (equivalent to 1,000 50% lethal doses) of the virulent S. typhimurium SVA44 became bacteremic with a profuse hemorrhagic diarrhea and had to be sacrificed within 2 to 7 days. The results suggest that the S. typhimurium antilipopolysaccharide immunity was insufficient to provide a solid protective efficacy against oral S. typhimurium infection. The immunohistopathological examination revealed that S. typhimurium SVA44 could be found in all layers of the intestinal mucosa and the lymphatic tissues of the Peyer's patches. In contrast, S. dublin SVA47 was found predominantly in the columnar enterocytes of the jejunum and ileum and the follicle-associated epithelium over the Peyer's patches. In addition, SVA47 was found in the glandular tissues of the duodenal and tonsillar areas and in the lungs. This suggests that the S. typhimurium and S. dublin strains have different virulence traits determining their tissue localization and dissemination.

Animals↗

Isotype-specific antibody responses of cattle to Salmonella Dublin lipopolysaccharide and porin following Salmonella Dublin vaccination and acute and chronic infection.

Stimulation of different T-cell subsets during antigen presentation influences the antibody isotype response to an antigen. Salmonella infection and Salmonella bacterin vaccination are likely to stimulate different T-cell subtypes. The objective of this study was to determine whether there are differences in the isotype response of cattle to Salmonella antigens following Salmonella infection and Salmonella bacterin vaccination. Sera from Salmonella bacterin-vaccinated, experimentally infected, and chronically infected (carrier) adult cattle collected during previous studies was used to evaluate the IgG1, IgG2, and IgM isotype responses of cows to Salmonella serotype Dublin lipopolysaccharide (LPS) and porin. Following vaccination and experimental oral infection, IgG1 titers to LPS and porin rose more quickly and persisted longer than did IgG2 titers. In contrast to Salmonella infection, bacterin vaccination stimulated a weak response to Salmonella porin. Salmonella infection also induced a higher IgG2:IgG1 titer ratio to LPS than did bacterin vaccination. Chronic Salmonella infection induced the highest LPS and porin IgG2:IgG1 titer ratios and the highest correlation between LPS and porin titers. Response operating characteristic curves for each isotype-specific enzyme-linked immunosorbent assay (ELISA) were determined to evaluate the effect of isotype on the sensitivity and specificity of Salmonella ELISA serology for distinguishing sera of Salmonella carriers from those of vaccinated and acutely infected cows. IgG2 titers to LPS and porin provide a more specific indicator of chronic Salmonella infection status than do IgG1 titers to the same antigens with little to no loss in sensitivity.

Acute Disease↗

Salmonella Dublin infection in Queensland dairy cattle.

OBJECTIVE: To investigate the presence of Salmonella Dublin in Queensland cattle. DESIGN: An epidemiological study using diagnostic laboratory information and farm records. PROCEDURE: Outbreaks of gastroenteritis or pneumonia in calves, and abortions and enteritis in cows were routinely investigated for the presence of salmonellae. Where S Dublin was isolated, attempts were made to gather further epidemiological information. RESULTS: Prior to 1983 only two outbreaks of S Dublin have been recorded in Queensland dairy cattle. In 1983 S Dublin abortions were diagnosed in dairy heifers introduced from southern Australia to south-east Queensland. Sampling indicated that at least 10% of the 500 introduced heifers were faecal excretors of S Dublin. On 3 of the 7 farms from which S Dublin was recorded, infection spread to other cattle that were in contact. From February 1985 to February 1996, 29 outbreaks of S Dublin in cattle occurred on 29 farms (28 in south east Queensland and 1 in north Queensland). Calves were primarily affected. Continuing outbreaks were confirmed on only 4 of these 29 farms. On 15 farms S Dublin infections were associated with the purchase of infected calves or cows, while another farm adjoined 2 previously infected farms. No source of S Dublin was evident for the other 13 farms, where histories were often inadequate. CONCLUSION: There has been a marked increase in S Dublin outbreaks in Queensland dairy cattle since 1983. Introduction of S Dublin carrier and aborting dairy heifers from southern Australia, where S Dublin is not uncommon, was associated with the initial outbreaks.

Animals↗

Evaluation and comparison of molecular techniques for epidemiological typing of Salmonella enterica subsp. enterica serovar dublin.

A total of 28 unrelated isolates of the Salmonella enterica subsp. enterica serovar dublin (S. dublin) collected during a 6-year period, as well as four samples of the S. dublin live vaccine strain Bovisaloral and its prototype strain S. dublin 442/039, were investigated by different molecular typing methods for the following reasons: (i) to find the most discriminatory method for the epidemiological typing of isolates belonging to this Salmonella serovar and (ii) to evaluate these methods for their capacity to discriminate among the live vaccine strain Bovisaloral, its prototype strain S. dublin 442/039, and field isolates of the serovar dublin. Five different plasmid profiles were observed; a virulence plasmid of 76 kbp as identified by hybridization with an spvB-spvC gene probe was present in all isolates. The detection of 16S rRNA genes and that of IS200 elements proved to be unsuitable for the epidemiological typing of S. dublin; only one hybridization pattern could be observed with each of these methods. The results obtained from macrorestriction analysis strongly depended on the choice of restriction enzyme. While the enzyme NotI yielded the lowest discriminatory index among all enzymes tested, it was the only enzyme that allowed discrimination between the Bovisaloral vaccine strain and its prototype strain. In contrast to the enzymes XbaI and SpeI, which only differentiated among the S. dublin field isolates, XhoI as well as AvrII also produced restriction fragment patterns of the Bovisaloral strain and of its prototype strain that were not shared by any of the S. dublin field isolates. Macrorestriction analysis proved to be the most discriminatory method not only for the epidemiological typing of S. dublin field isolates but also for the identification of the S. dublin live vaccine strain Bovisaloral.

Animals↗

Immunizing efficacy of aromatic-dependent Salmonella dublin in mice and calves.

Mice immunized with an aromatic-dependent (aro-) S. dublin strain CS101 by either the intraperitoneal (i.p.) or oral route, were protected against oral challenge with a virulent S. dublin strain CS90, the degree of protection being the greatest when mice had received 3 immunizing doses at weekly intervals. Mice immunized with an aromatic-dependent (aro-) S. typhimurium strain CS332 by the i.p. or oral routes were protected against challenge with virulent S. dublin strain CS90 at 1 or 2 weeks but not at 3 or 4 weeks post-immunization. Mice immunized with 1 dose of aro- S. dublin strain CS101 by the i.p. route developed low levels of lipopolysaccharide (LPS) and flagellin-specific antibody but no delayed-type hypersensitivity (DTH) whereas those immunized with 2 or 3 doses developed significantly higher antibody titres and DTH. In contrast, mice immunized by the oral route developed neither significant antibody response nor DTH. The aro- S. dublin strain CS101 could not be detected beyond day 28 post-inoculation in visceral organs including liver, spleen, mesentery, small intestine, caecum or large intestine of mice inoculated by the i.p. route or in mice inoculated by the oral route with the exception of day 42 post-inoculation. Challenge of mice previously immunized with 3 doses of the aro- S. dublin strain CS101 by the i.p. or oral route with virulent S. dublin strain CS90 resulted in their rapid clearance from the above visceral organs. Calves immunized with the aro- S. dublin strain CS101 by either the intramuscular (i.m.) or oral routes were significantly protected against oral challenge with virulent S. dublin strain CS90. In contrast to the observations in mice, somatic (O) and flagellar (H) antibody titres of calves immunized by either route were negligible as were anti-LPS antibody titres. However, flagellin-specific antibody titres were higher in calves immunized by the i.m. than the oral route. These results indicate that the protection observed in immunized mice or calves against oral challenge with virulent S. dublin was unlikely to have been mediated by humoral salmonella-specific immune mechanism(s).

Administration, Oral↗

Salmonella dublin infection of calves: use of small doses to simulate natural infection on the farm.

Small numbers of Salmonella dublin were used to infect calves in an attempt to simulate natural infection on the farm. Twenty calves were exposed to S. dublin by one or more of the following methods: Sucking cows which were excreting S. dublin in their faeces (less than 10(2)-10(5) organisms/g). Housing on S. dublin contaminated bedding. Drinking S. dublin contaminated water (10(2)-10(4) organisms/ml). During this experiment some calves were given therapeutic does of oxytetracycline. After exposure the calves were examined for faecal excretion of S. dublin (in some instances mouth swabs and blood samples were also examined) and for clinical signs of illness. Most of the calves became infected with S. dublin but excretion was usually sporadic and the numbers of salmonellas excreted were small. No clinical signs of salmonellosis were observed by S. dublin was isolated from one calf at post-mortem. Another six calves, dosed orally with either 10(6) or 10(8) S. dublin, showed signs of mild illness and although three calves had diarrhoea excretion of salmonellas was intermittent. S. dublin was isolated from one of these calves at post-mortem.

Animals↗

Persistent experimental Salmonella dublin intramammary infection in dairy cows.

Experimental intramammary infections were induced in five post-parturient Holstein cows by inoculation of low numbers (5000 colony forming units) of virulent Salmonella dublin via the teat canal of mammary gland quarters. Rectal temperature, pulse and respiratory rates, milk yield, and milk quality as assessed by the California Mastitis Test (CMT) and somatic cell counts (SCC) were recorded every 12 hours at milking. Bacteriologic cultures of foremilk quarter samples and feces were obtained daily, as were complete blood counts. ELISA titers for IgG and IgM recognizing S. dublin lipopolysaccharide (LPS) were obtained weekly on serum and quarter milk samples. All cows excreted S. dublin intermittently from infected quarters, but no changes were detected in rectal temperature, appearance of the mammary gland or secretions, CBC, milk yield, and pulse and respiratory rates. Somatic cell counts were modestly increased in infected quarters as compared with uninfected quarters (P = .015, paired t test); however, CMT scores after infection remained low, and were not significantly different from pre-infection scores (P greater than .10, sign test). After infection, administration of dexamethasone resulted in signs of clinical mastitis and increased excretion of S. dublin from mammary quarters (P = .0004, paired t test). One cow had necrotizing mastitis and S. dublin septicemia and was euthanatized. In the four surviving cows, clinical improvement was observed after systemic gentamicin therapy and intramammary infusion with polymyxin B, but all cows continued to excrete S. dublin intermittently from one or more quarters and occasionally from feces for the remaining period of observation. All infected cows demonstrated a rise in IgG and IgM ELISA titers recognizing S. dublin LPS in serum and milk. At necropsy (13-25 weeks postinfection), S. dublin was recovered only from the mammary tissue or supramammary lymph nodes in three of four cows. In one cow, mammary gland and lymph-node samples were negative for S. dublin despite positive milk cultures. In all cows, histopathologic examination revealed multifocal areas of chronic active mastitis. These lesions were similar to histopathologic findings from mammary gland carriers with naturally acquired S. dublin infection.

Animals↗

Use of ELISA for detection of immunoglobulins G and M that recognize Salmonella dublin lipopolysaccharide for prediction of carrier status in cattle.

Immunoglobulin reactions to Salmonella dublin in serum and milk from 4 groups of lactating cows were measured by an indirect ELISA. The groups consisted of (1) cows that were natural carriers of S dublin in the mammary gland, (2) experimentally infected cows that did not become carriers, (3) cows inoculated with a commercial S dublin bacterin, and (4) cows used as S dublin-negative controls. Milk and serum samples were obtained at monthly intervals. Models for predicting carrier status were developed by use of stepwise logistic regression. Independent variables consisted of serum and milk IgG and IgM titers to S dublin lipopolysaccharide and a ratio of IgG to IgM. The utility of a single sample vs multiple samples obtained at 1-month or 2-month intervals was tested by comparison of goodness-of-fit chi 2 P values for 8 models predicting carrier status. Immunoglobulin reactions specific to S dublin were a significant predictor of carrier status (P less than 0.001). Serum IgG titers specific for S dublin were the most important variable for predicting carrier status. Two serum IgG titers to S dublin obtained 2 months apart was a better predictor of carrier status than measurement of the IgG:IgM ratio from a single serum sample. Immunoglobulin recognizing S dublin epitopes also were detected in milk samples. In milk, performing 2 ELISA 60 days apart to determine IgG and IgM reactions to S dublin appeared to be useful for the prediction of carrier status, but was not as accurate as models for serum immunoglobulin reactions.

Animals↗

Salmonella enterica serovar Dublin strains which are Vi antigen-positive use type IVB pili for bacterial self-association and human intestinal cell entry.

Some strains of Salmonella enterica serovar Dublin are Vi antigen-positive. S. enterica serovar Typhi uses Type IVB pili, encoded adjacent to the viaB locus required for Vi antigen synthesis, to facilitate both eukaryotic cell attachment and bacterial self-association under conditions that favour DNA supercoiling. These pilus-mediated events may be important in typhoid fever pathogenesis. A survey of 17 isolates of S. enterica serovar Dublin showed that all strains which carried the viaB region also carried a serovar Typhi-like Type IVB pil operon, and all serovar Dublin Vi antigen-negative isolates lacked the pil operon. The pil operon was completely sequenced from one of the Vi(+) serovar Dublin strains, and was almost identical (4 nt changes; 3 aa changes, in over 10 kb) to that of serovar Typhi. A pilS mutant of one serovar Dublin strain was constructed, and shown to invade cultured human intestinal INT407 cells to an extent only 20% that of the wild-type parent. Purified prePilS protein inhibited INT407 cell entry by serovar Dublin. The wild-type serovar Dublin strain, but not the pilS mutant, self-associated. The data suggest that the serovar Dublin Type IVB pil operon may increase the human-invasiveness of serovar Dublin, compared to pil-free strains.

Antigens, Bacterial↗

Evaluation of three newly developed enzyme-linked immunosorbent assays and two agglutination tests for detecting Salmonella enterica subsp. enterica serovar dublin infections in dairy cattle.

In this study test characteristics of three newly developed enzyme-linked immunosorbent assays (ELISAs) for Salmonella enterica subsp. enterica serovar Dublin were evaluated and compared with two agglutination tests. The ELISAs involved were an indirect ELISA with serovar Dublin lipopolysaccharide (LPS ELISA), an indirect ELISA with serovar Dublin flagellar antigen (GP ELISA), and a double-antibody sandwich blocking ELISA that uses monoclonal antibodies against S. enterica subsp. enterica serovar Enteritidis flagellin (GM-DAS ELISA). The agglutination tests involved were two routine serum agglutination tests with either somatic (O) or flagellar (H) antigen. Diagnostic specificity of the three ELISAs was determined using 840 serum samples from seven dairy herds without any history of serovar Dublin infection. Cutoff values at a titer of 100, 100, and 10, respectively, for the LPS ELISA, GP ELISA, and GM-DAS blocking ELISA resulted in a specificity of 99.3, 100, and 100%, respectively. Using these cutoff values the LPS ELISA, GP ELISA, and GM-DAS ELISA were able to detect, respectively, 30, 46, and 38% of 50 fecal culture-positive animals from 13 herds with a recent serovar Dublin infection. With the same cutoff values, active carriers (n = 18) were detected for 94.4% with the LPS ELISA and for 100% with the GP and GM-DAS ELISAs. Kappa values determined on the results of all tests from 8 of the 13 serovar Dublin-infected herds and the 7 control herds demonstrated a good correlation between the results of all ELISAs and the H-agglutination test. The results of the O-agglutination test failed to correlate with those of the other tests. Using a set of sera from 170 aborting cows (with 25 abortions due to serovar Dublin), test results of the ELISAs and the H-agglutination test were comparable. The H-agglutination test may be used successfully for single sample testing, especially to diagnose abortion due to serovar Dublin. It is concluded that the ELISAs are useful diagnostic tools in serovar Dublin control programs and that they are preferred to agglutination tests for reasons of automation and costs.

Abortion, Veterinary↗

Antibody response and protection against challenge in mice vaccinated intraperitoneally with a live aroA O4-O9 hybrid Salmonella dublin strain.

An auxotrophic Salmonella dublin (O9,12) strain, SL5631, with a deletion affecting gene aroA, was made into a partial diploid expressing the rfb (O-antigen-repeat-unit-specifying) gene cluster of Salmonella typhimurium (O4,12). By use of O4- and O9-specific antisera in indirect immunofluorescence assays, the resulting hybrid SL7103 was shown to express both the O4- and O9-antigen epitopes in the same bacterium. Qualitative and quantitative sugar analyses by gas-liquid chromatography on peralditol acetates of phenol-water-extracted lipopolysaccharides showed that the S. dublin and S. typhimurium repeating units (estimated on the basis of their tyvelose and abequose contents, respectively) were present in approximately equimolar amounts. The SL7103 hybrid auxotroph was avirulent when given intraperitoneally to NMRI mice in a dose of 10(8) CFU and elicited a protective immunity against intraperitoneal challenge with either virulent S. dublin (50% lethal dose of ca. 1.5 x 10(4) CFU versus < 1 x 10(1) CFU in nonimmunized mice) or virulent S. typhimurium (50% lethal dose of ca. 1 x 10(5) versus < 1 x 10(1) CFU in nonimmunized mice). Compared with the protection elicited in homologous systems (S. dublin SL5631 against S. dublin and S. typhimurium SL1479 against S. typhimurium), the protective efficacy of the hybrid was reduced approximately 70-fold against S. dublin challenge and 100-fold against S. typhimurium challenge. Vaccination with S. typhimurium SL1479 conferred no protection against S. dublin challenge, and vaccination with S. dublin SL5631 conferred no protection against S. typhimurium challenge. The protection elicited by the hybrid strain SL7103 is supposed to be mainly a consequence of serum antibodies directed against the immunodominant O4 and O9 epitopes.

Animals↗

Interaction of Salmonella choleraesuis, Salmonella dublin and Salmonella typhimurium with porcine and bovine terminal ileum in vivo.

Quantitative experiments on the interaction of Salmonella choleraesuis and Salmonella dublin with porcine and bovine intestinal epithelia yielded no evidence to suggest that host restriction of S. choleraesuis and S. dublin for pigs and calves respectively could be explained in terms of the patterns of intestinal invasion observed in ligated ileal loops in vivo, at 3 h after challenge. No evidence was found to support the idea that Peyer's patches, or specifically M cells, are the major route of entry for these serotypes in vivo. Three hours after loop inoculation, each serotype was recovered in comparable numbers from either absorptive or Peyer's patch mucosae present in the same ileal loop, indicating that both types of tissue are involved in the early stages of the enteropathogenic process induced by both serotypes. More detailed transmission electron microscopic (TEM) analyses of follicle-associated epithelia (FAE) challenged with S. choleraesuis showed that in the same region of FAE, organisms invaded both M cells and enterocytes directly; comparable detailed TEM studies with S. dublin could not be carried out because of the tissue-destructive properties of this serotype. S. dublin was clearly more histotoxic than S. choleraesuis as had previously been found in rabbits: this difference is almost certainly due to a tissue-damaging toxin which is neither host nor gut-tissue specific. The tissue-destructive potential of S. dublin has profound implications for the measurement of and the assignment of significance to the invasiveness of S. dublin. S. dublin was nearly always seen entering gut cells in micro-colonies whereas S. choleraesuis entered mainly as single organisms or small groups of two or three.

Animals↗

Effects of Fasciola hepatica infection on responses of rats to reinfection with Salmonella dublin.

The survival rate of rats given potentially lethal doses of 7.6 X 10(8) to 10(9) Salmonella dublin intraperitoneally was enhanced in those which had been infected six weeks previously with 10(2) S dublin by the same route. Fasciola hepatica given six weeks before or one week after 10(2) S dublin did not alter the survival rate of rats reinfected with S dublin. However high levels (10(4) to 10(6) per g) of S dublin persisted in the tissues and faeces of fluke-infected but not of fluke-free rats. Agglutinating antibody responses to S dublin were unimpaired in fluke-infected rats but cutaneous delayed hypersensitivity reactions were slightly reduced. the humoral agglutinating antibody response may be important for survival of the host but other responses may be necessary for elimination of S dublin from the tissues.

Agglutinins↗

Safety and efficacy of an avirulent live Salmonella choleraesuis vaccine for protection of calves against S dublin infection.

OBJECTIVE: To evaluate the safety and efficacy of avirulent live Salmonella choleraesuis strain 54 (SC54) as a vaccine to protect calves against salmonellosis caused by S dublin. ANIMALS: 40 head of clinically normal 3 to 5-week-old male Holstein calves that were culture negative for Salmonella sp. PROCEDURE: Calves were randomly assigned to 4 test groups of 10 calves each. Group 1 received 8.5 x 10(7) colony-forming units (CFU) of SC54 SC. Groups 2 and 3 received 1.13 x 10(9) CFU of SC54, SC and intranasally, respectively. Group 4 received saline solution as a vaccine control. All calves were challenge exposed orally with 1.74 x 10(9) CFU of virulent S dublin 14 days after vaccination. Clinical signs and Salmonella shedding were monitored for 28 days after vaccination. Calves were necropsied, and organs were cultured for Salmonella sp 14 days after challenge exposure. RESULTS: Calves of groups 2 and 3 had slightly high rectal temperature after vaccination. Salmonella dublin challenge exposure resulted in mild clinical signs of salmonellosis. All vaccinated groups had significantly (P < 0.05) lower rectal temperature, fecal shedding of S dublin, and recovery of S dublin from organs after necropsy. SC54 was not recovered from fecal or blood samples collected after vaccination or from injection site samples or organs collected at necropsy. CONCLUSIONS: SC54 given intranasally or SC to calves was safe and significantly (P < 0.05) reduced clinical signs and bacterial shedding after oral challenge exposure with S dublin. CLINICAL RELEVANCE: SC54 has potential as an effective vaccine to aid in prevention of salmonellosis caused by S dublin in calves.

Administration, Intranasal↗